I. Field of the Invention
The present invention relates to the fields of molecular biology and medicine. More particularly, it relates to zinc finger nucleases and their use in treating cystic fibrosis.
II. Related Art
A. Cystic Fibrosis
Cystic fibrosis, or CF, is an inherited disease of epithelia-lined organs, including the glands that make mucus and sweat. “Inherited” means that the disease is passed through the genes from parents to children. People who have CF inherit two faulty cystic fibrosis transmembrane conductance regulator (CFTR) genes, one from each parent. CF mostly affects the lungs, pancreas, liver, intestines, sinuses, and sex organs. The symptoms and severity of CF vary from person to person—some people who have CF have serious lung and digestive problems, while others have more mild disease that does not show up until they are adolescents or adults. The symptoms and severity of CF also vary over time.
The disease manifests because if one has CF, mucus becomes thick and sticky. In the lungs, airway innate immunity is impaired, mucus builds up and blocks airways. The buildup of mucus makes it easy for bacteria to grow, which leads to repeated, serious lung infections. Over time, these infections can severely damage lungs. The abnormal secretions also can block ducts in the pancreas. As a result, the digestive enzymes that your pancreas makes cannot reach your small intestine, causing vitamin deficiency and malnutrition because nutrients leave the body unused. It also can cause bulky stools, intestinal gas, a swollen belly from partial or complete intestinal obstruction, and pain or discomfort. CF also causes your sweat to become very salty and, as a result, your body loses large amounts of salt when you sweat. This can upset the balance of minerals in your blood and cause a number of health problem, including dehydration, increased heart rate, tiredness, weakness, decreased blood pressure, heat stroke, and, rarely, death.
As treatments for CF continue to improve, so does life expectancy for those who have the disease. Today, some people who have CF are living into their thirties, forties, fifties, or older. However, CF remains the leading genetic cause of premature death in the United States. As such, improved methods of treating CF are in great need.
B. Zinc Finger Nucleases
Zinc fingers are among the most common DNA binding motifs found in eukaryotes. It is estimated that there are 500 zinc finger proteins encoded by the yeast genome and that perhaps 1% of all mammalian genes encode zinc finger containing proteins. These proteins are classified according to the number and position of the cysteine and histidine residues available for zinc coordination.
The CCHH class, typified by the Xenopus transcription factor IIIA, is the largest. These proteins contain two or more fingers in tandem repeats. In contrast, the steroid receptors contain only cysteine residues that form two types of zinc-coordinated structures with four (C4) and five (C5) cysteines. Another class of zinc fingers contains the CCHC fingers. The CCHC fingers, which are found in Drosophila, and in mammalian and retroviral proteins, display the consensus sequence C—N2—C—N4—H—N4—C (SEQ ID NO:111). Recently, a novel configuration of CCHC finger, of the C—N5—C—N12—H—N4—C (SEQ ID NO:112) type, was found in the neural zinc finger factor/myelin transcription factor family. Finally, several yeast transcription factors such as GAL4 and CHA4 contain an atypical C6 zinc finger structure that coordinates two zinc ions. Zinc fingers are usually found in multiple copies (up to 37) per protein. These copies can be organized in a tandem array, forming a single cluster or multiple clusters, or they can be dispersed throughout the protein.
Zinc finger nucleases (ZFNs) can be used to “rewrite” the sequence of an allele by invoking the homologous recombination machinery to repair the double-strand breaks using a supplied DNA fragment as a template. The homologous recombination machinery searches for homology between the damaged chromosome and the extra-chromosomal fragment and copies the sequence of the fragment between the two broken ends of the chromosome, regardless of whether the fragment contains the original sequence. If the subject is homozygous for the target allele, the efficiency of the technique is reduced since the undamaged copy of the allele may be used as a template for repair instead of the supplied fragment.
Custom-designed ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand breaks to the genome, and stimulate local homologous recombination by several orders of magnitude. This makes targeted gene correction or genome editing a viable option in human cells. Since ZFN-encoded plasmids can be used to transiently express ZFNs to target a double-strand break to a specific gene locus in human cells, they offer an excellent way for targeted delivery of the therapeutic genes to a pre-selected chromosomal site. The ZFN-encoded plasmid-based approach has the potential to circumvent problems associated with viral delivery of therapeutic genes. Alternatively, ZFN pairs can be packaged in a variety of viral vectors to improve delivery to specific cell types.